Methods and compositions for screening for presynaptic calcium channel blockers

Abstract

Methods and compositions related to the identification of compounds that block neurotransmitter release are disclosed. Using the methods of the present invention, candidate compounds may be screened for the ability to bind to presynaptic calcium channels such that the docking of presynaptic vesicles to presynaptic calcium channels will be inhibited. The present invention also discloses peptides useful in the screening methods.

We claim: 
     
       1. A peptide consisting of the amino acid sequence of FIG. 11A from alanine, amino acid 773, to aspartic acid, amino acid 859. 
     
     
       2. A peptide consisting of the amino acid sequence of FIG. 11A from glutamic acid, amino acid 718, to aspartic acid, amino acid 859. 
     
     
       3. A peptide consisting of the amino acid sequence of FIG. 11A from glutamic acid, amino acid 718, to cysteine, amino acid 1141. 
     
     
       4. A peptide consisting of the amino acid sequence of FIG. 11A from an amino acid positioned between glutamic acid, amino acid 718, and alanine, amino acid 773, to an amino acid positioned between aspartic acid, amino acid 859, and cysteine, amino acid 1141. 
     
     
       5. A peptide consisting of an amino acid sequence of between 87 to 424 amino acid residues in length, wherein said amino acid sequence contains an amino acid sequence having at least 60% sequence similarity with the amino acid sequence of FIG. 11A from alanine, amino acid 773, to aspartic acid, amino acid 859. 
     
     
       6. A peptide consisting of the amino acid sequence of FIG. 11B from alanine, amino acid 772, to aspartic acid, amino acid 858. 
     
     
       7. A peptide consisting of the amino acid sequence of FIG. 11B from glutamic acid, amino acid 717, to aspartic acid, amino acid 858. 
     
     
       8. A peptide consisting of the amino acid sequence of FIG. 11B from glutamic acid, amino acid 717, to cysteine, amino acid 1143. 
     
     
       9. A peptide consisting of the amino acid sequence of FIG. 11B from an amino acid positioned between glutamic acid, amino acid 717, and alanine, amino acid 772, to an amino acid positioned between aspartic acid, amino acid 858, and cysteine, amino acid 1143. 
     
     
       10. A peptide consisting of an amino acid sequence of between 87 to 427 amino acid residues in length, wherein said amino acid sequence contains an amino acid sequence having at least 60% sequence similarity with the amino acid sequence of FIG. 11B from alanine, amino acid 772, to aspartic acid, amino acid 858. 
     
     
       11. A peptide consisting of the amino acid sequence of FIG. 12 from isoleucine, amino acid 181, to glycine, amino acid 288. 
     
     
       12. A method of screening for compounds that inhibit the interaction between presynaptic calcium channels and presynaptic vesicles, comprising the steps of: (a) contacting a peptide according to any one of claims 1-10 with a candidate compound under conditions sufficient to permit binding between said peptide and said candidate compound; and   (b) detecting the presence or absence of binding between said peptide and said candidate compound, thereby determining whether said candidate compound bound to said peptide.   
     
     
       13. A method of screening for compounds that inhibit the interaction between presynaptic calcium channels and presynaptic vesicles, comprising the steps of: (a) incubating a candidate compound, a first peptide according to any one of claims 1-10, and a second peptide consisting of the amino acid sequence of FIG. 12 from isoleucine, amino acid 181, to glycine, amino acid 288, under conditions sufficient to permit binding between said first and second peptides; and   (b) detecting the presence or absence of binding between said first and second peptides, thereby determining whether said candidate compound inhibited the binding.   
     
     
       14. A method of screening for compounds that inhibit the interaction between presynaptic calcium channels and presynaptic vesicles, comprising the steps of: (a) incubating a first peptide according to any one of claims 1-10 and a candidate compound under conditions sufficient to permit binding between said peptide and said candidate compound, to form a reaction mixture;   (b) contacting a second peptide consisting of the amino acid sequence of FIG. 12 from isoleucine, amino acid 181, to glycine, amino acid 288, with said reaction mixture under conditions sufficient to permit binding between said first and second peptides; and   (c) detecting the presence or absence of binding between said first and second peptides, thereby determining whether said candidate compound inhibited the binding.   
     
     
       15. A method of screening for compounds that inhibit the interaction between presynaptic calcium channels and presynaptic vesicles, comprising the steps of: (a) incubating a candidate compound, a peptide according to any one of claims 1-10, and syntaxin under conditions sufficient to permit binding between said peptide and said syntaxin; and   (b) detecting the presence or absence of binding between said peptide and said syntaxin, thereby determining whether said candidate compound inhibited the binding.   
     
     
       16. A method of screening for compounds that inhibit the interaction between presynaptic calcium channels and presynaptic vesicles, comprising the steps of: (a) incubating a peptide according to any one of claims 1-10 and a candidate compound under conditions sufficient to permit binding between said peptide and said candidate compound, to form a reaction mixture;   (b) contacting syntaxin with said reaction mixture under conditions sufficient to permit binding between said peptide and said syntaxin; and   (c) detecting the presence or absence of binding between said peptide and said syntaxin, thereby determining whether said candidate compound inhibited the binding.